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. 2023 Aug 29;26(10):107780. doi: 10.1016/j.isci.2023.107780

Figure 2.

Figure 2

Regional differences in mitochondrial network connectivity, content and localization of ONC-responsive IPL and OPL microglia

(A) Schematic of optic nerve crush (ONC). Forceps induce unilateral damage (red) to axonal projections (orange) of retinal ganglion cells (RGCs) posterior to the optic disc. Right, retinal side-view schematic. OPL, outer plexiform layer. IPL, inner plexiform layer. GCL, ganglion cell layer.

(B) Confocal images of RBPMS-immunostained naive WT retinas or 5 days after ONC WT co-labeled with cleaved caspase-3 (CASP3, red) to identify dying retinal ganglion cells (RGCs, white). Right, bar plot indicate number of caspase-3+/RBPMS+ cells. Wilcoxon rank-sum test: p = 0.0016. Scale bar: 50 μm.

(C) Overview confocal image of immunostained IPL microglia (IBA1, magenta) in naive WT (left) and 5 days after ONC WT (right) condition. Scale bar: 30 μm.

(D–M) Comparison of mitochondrial response in naive (magenta border) microglia WT (gray) and 5 days after ONC WT (blue). Boxplot minimum and maximum: InterQuartile Range (IQR) around median (center line). Whiskers: 1.5 IQRs. Black diamond: outliers outside of 1.5 IQRs. (D–H) Mitochondrial parameters for IPL microglia. (D–E) Mito-Dendra2 (green) expression in the IBA1-immunostained IPL microglia for naive WT microglia (D) or ONC WT microglia (E). Next, corresponding 3D-surfaces. Below: Zoom-in of region of interest (dashed line) from image and 3D-surface. Scale bar: 10 μm, zoom-in: 3 μm. (F) Mitochondrial network connectivity determined by median mitochondrial volume (left, Welch’s t-test: p < 0.0001), number of organelles within a single cell (center, Wilcoxon rank-sum test: p = 0.0018) and mean mitochondrial sphericity (right, T-test: p < 0.0001). (G) Percentage of mitochondrial volume per microglial volume. T-test: p = 0.0040. (H) Mitochondrial localization. Scatterplot depicting mitochondria volume vs. distance from the cell soma (0, origin) of the population of mitochondria in naive WT (left) or ONC WT (right) microglia. Point density, pseudo-colored. Percentage of total mitochondrial volume localized within the perinuclear region. Wilcoxon rank-sum test: p < 0.0001. (I–M) Mitochondrial parameters for OPL microglia. (I–J) Representative IBA1-immunostained OPL microglia (magenta) expressing mito-Dendra2 (green) from naive WT (I) or ONC WT microglia (J). Next, corresponding 3D-surfaces. Below: Zoom-in of region of interest (dashed line) from image and 3D-surface. Scale bar: 10 μm, zoom-in: 3 μm. (K) Mitochondrial network connectivity determined by median mitochondrial volume (left, Student’s t test: p = 0.0026), number of organelles within a single cell (center, Student’s t test: p = 0.0900) and mean mitochondrial sphericity (Student’s t test, p = 0.79389). (L) Percentage of mitochondrial volume per microglial volume. Student’s t test: p = 0.8441. (M) Mitochondrial localization. Scatterplot depicting mitochondria volume vs. distance from the cell soma (0, origin) of the population of mitochondria in naive WT (left) or ONC WT (right) microglia. Point density, pseudo-colored. Percentage of total mitochondrial volume localized within the perinuclear region. Welch’s t-test: p = 0.0045. ∗∗p < 0.01, ∗∗∗p < 0.001, n.s. p > 0.05: not significant. See Table S2 for retina and cell numbers, statistical tests and corresponding data.